Project Summary This Translational Science Acceleration Supplement application is being submitted to greatly accelerate our discovery of mechanisms underlying biliary atresia (BA) pathogenesis and biomarker discovery for mitochondrial hepatopathies (MH), by using translational approaches and biospecimens already obtained in ChiLDReN studies. In the IVIG clinical trial (PRIME) in BA in ChiLDReN, it was discovered that plasma IL-8 (a neutrophil chemokine) and neutrophil extracellular trap (NET) formation correlated with rising serum bilirubin at 90 days post-Kasai, and that increasing levels of IL-8 in the first 60 days post-Kasai was associated with significantly increased risk of liver transplant/ death at 1 year. This led to our hypothesis that the immunopathogenesis of BA involves HMGB1-induced neutrophil activation, resulting in NET formation and NET-associated stellate cell activation. This will be tested by the following aims using ChiLDReN plasma, fixed liver tissue and peripheral blood neutrophils from PROBE BA patients and controls: (1) establish the mechanism of NETosis in BA: and (2) determine if NET-induced hepatic stellate cell activation occurs in BA. We anticipate finding that NETosis will be increased in BA, will be NOX2 dependent, and will promote hepatic stellate cell activation, which will establish a role for neutrophils in BA progression. In a second project, we will investigate potential metabolomic and proteomic biomarkers for MH. MH may present as acute liver failure, chronic liver disease, fatty liver disease, cholestasis or cirrhosis and may be difficult to distinguish from other etiologies. There are no simple tests to detect the presence of a MH in an individual patient, nor are there predictive or surrogate biomarkers for these disorders. The main objective of this proposal is to evaluate plasma/serum samples from MITOHEP study participants and disease controls (from PROBE, BASIC and LOGIC), for metabolomic (candidate and discovery) and protein (candidate) signatures that would be specific and sensitive for MH. This study will be a collaboration between investigators at University of Colorado Denver and Massachusetts General Hospital in Boston. Aims of this study are: (1) determine if a recently identified plasma metabolomic biomarker panel present in adults with the mitochondrial disease MELAS would distinguish MITOHEP participants from alpha-1 antitrypsin deficiency; (2) perform discovery metabolomics to identify novel metabolite signatures for MH; and (3) determine the clinical utility of the candidate protein mitochondrial biomarkers, fibroblast growth factor 21 (FGF-21) and growth differentiation factor 15 (GDF15), in MITOHEP participants, liver disease controls and healthy pediatric controls. We anticipate that the metabolite panel and protein biomarkers will distinguish MITOHEP patients from the disease control groups as well as normal controls, and that we may discover new metabolite biomarkers for the diseases studied. If true, follow on studies would need to determine if these biomarkers correlate with other markers of liver function, are predictive of outcomes, and if the biomarkers normalize after liver transplantation.